Updated project metadata. The colonic epithelium is a highly dynamic system important for regulation of ion and water homeostasis via absorption and secretion, and in maintaining a protective barrier between the outer milieu and the inside of our body. These processes are known to gradually change along the length of the colon, although a complete characterization at the protein level is lacking. We therefore analyzed the membrane proteome of isolated human (n=4) colonic epithelial cells from biopsies obtained via routine colonoscopy for four segments along the large intestine: ascending, transverse, descending and sigmoid colon. Label-free quantitative proteomic analyses using high-resolution mass spectrometry were performed on enriched membrane proteins. The results showed a stable level for the majority of membrane proteins, while a distinct decrease in proteins associated with bacterial sensing, cation-transport and O-glycosylation in the proximal to distal direction. Proteins involved in microbial defense and anion-transport showed on the other hand an opposing gradient and increased towards the distal end. The gradient of ion-transporter proteins could be directly related to previously observed ion transport activities. All individual glycosyltransferases required for the O-glycosylation of the major colonic mucin MUC2 were observed and correlated with the known glycosylation variation along the colon axis. This is the first comprehensive quantitative dataset of membrane protein abundance along the human colon and will add to the knowledge of the physiological function of the different regions of the colonic mucosa.